Apparatus for the transfer of bio-ink

Abstract

An apparatus for transferring bio-ink onto a target having a slide defining a receiving area of a film of fluid containing inhomogeneities, a laser source associated with controlled diversion means and an optical block for focusing in a plane of the fluid film in order to apply a local pulse, wherein the apparatus also comprises imaging means and means for analyzing images in order to recognize the geometric positions of the inhomogeneities in the film, and an observable feature of each of the inhomogeneities (size, shape factor, type of particles, age of the particle, density, type of biomaterial, molecule, etc.) recognized by the appropriate analysis means. The apparatus further comprises selection means for selecting at least one of the inhomogeneous areas, and means for controlling the diversion in order to direct the laser beam toward the position of the inhomogeneous area and trigger the firing of the laser.

Claims

1. An apparatus for transferring bio-ink to a target, comprising: a slide defining a reception area for a fluid film containing heterogeneities; a laser associated with a controlled deflection device and an optical unit for focusing a laser beam emitted by the laser in a plane of the fluid film to apply a localized laser pulse, the slide being transparent to the laser beam; an imager located and configured to acquire an image of an imaging area of the fluid film, the imaging area being at least five times larger than a nominal cross-section of an inhomogeneity of the fluid film, the slide being transparent in the spectral domain of the imager; a computer configured to analyze the acquired image and recognize geometric positions of inhomogeneities in the fluid film and an observable characteristic of each of the recognized inhomogeneities; and wherein the apparatus is configured to select at least one of the recognized inhomogeneous zones, and control the deflection device to direct the laser beam toward the position of the inhomogeneous zone and trigger the localized laser pulse.

2. The apparatus of claim 1, wherein the inhomogeneities of the fluid film comprise particles.

3. The apparatus of claim 1, wherein the inhomogeneities of the fluid film comprise biomaterials.

4. The apparatus of claim 1, wherein the inhomogeneities of the fluid film comprise biochemical species.

5. The apparatus of claim 1, wherein the inhomogeneities of the fluid film comprise a combination of particles and/or biomaterials and/or biochemical species.

6. The apparatus of claim 1, wherein the imaging area is greater than one square millimeter.

7. The apparatus of claim 1, wherein the imaging area includes an area of interaction between the laser beam and the fluid film.

8. The apparatus of claim 1, wherein the imaging area is distinct from an interaction area between the laser beam and the fluid film.

9. The apparatus of claim 1, wherein the computer is remote from the laser, imager, and slide, the apparatus further comprising means of communication between the imager and the computer.

10. The apparatus of claim 1, further comprising a spectrometer.

11. The apparatus of claim 1, wherein the fluid film comprises living cells.

12. The apparatus of claim 1, wherein the apparatus is configured to form more than one area of interaction between laser beams and the fluid film.

13. The apparatus of claim 1, further comprising a suction system for removing excess printed material from a printing area.

14. The apparatus of claim 1, further comprising means for imaging a printed substrate.

15. The apparatus of claim 1, wherein the imager is configured to acquire images in real time.

16. A method for transferring bio-ink to a target, comprising: placing in a transfer equipment a transparent slide defining a reception area for a transparent fluid film containing a plurality of inhomogeneities; at least one imaging step of acquiring an image of an area of the film, the image having an image cross-section at least five times larger than a nominal cross-section of an inhomogeneity; at least one image analysis step of locating geometric positions of the inhomogeneities and characterizing at least one observable characteristic of each of the located inhomogeneities; at least one selection step of selecting one of the located and characterized inhomogeneities; and at least one transfer step comprising controlling an orientation and activation of a laser pulse emitted by a laser associated with a controlled deflection device and directing the laser beam to the position of one inhomogeneity and triggering a laser pulse and interaction of the laser beam with the film at the location of the one inhomogeneity.

17. The method of claim 16, wherein the inhomogeneities comprise particles.

18. The method of claim 16, wherein the inhomogeneities comprise biomaterials.

19. The method of claim 16, wherein the inhomogeneities comprise biochemical species.

20. The method of claim 16, wherein the inhomogeneities comprise a combination of particles, and/or biomaterials and/or chemical species.

21. The method of claim 16, wherein the at least one transfer step comprises a plurality of transfer steps each corresponding to at least one localized and characterized inhomogeneity.

22. The method of claim 16, wherein the at least one transfer step is performed after the at least one imaging step, the at least one image analysis step, and the at least one selection step.

23. The method of claim 16, further comprising a step of calculating an ordered sequence of inhomogeneities to be transferred.

24. The method of claim 16, wherein the at least one imaging step comprises processing an acquired image to extract simplified patterns corresponding to imaged inhomogeneities.

25. The method of claim 16, wherein the locating of the geometric positions of the inhomogeneities and the characterizing of the at least one observable characteristic of each of the inhomogeneities are carried out simultaneously.

26. The method of claim 16, wherein the locating of the geometric positions of the inhomogeneities and the characterizing of the at least one observable characteristic of each of the plurality of inhomogeneities are performed by processing by algorithms executed on processors with parallel architecture.

27. The method of claim 16, further comprising a step of comparing an acquired image of a transfer area after the at least one transfer step with a map of an intended implantation.

28. The method of claim 27, further comprising an additional step of triggering another at least one transfer step to correct a difference between the acquired image and the map of the intended implantation.

29. The method of claim 27, further comprising a suction step to correct a difference between the acquired image and the map of the intended implantation.

30. The method of claim 16, wherein the transparent fluid film comprises living cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other characteristics and advantages will appear from the following description of the present disclosure, the description being given by way of example only, with reference to the appended drawings in which:

(2) FIG. 1 is a schematic view of an equipment according to the present disclosure.

(3) FIG. 2 shows views of the acquired images.

DETAILED DESCRIPTION

(4) The present disclosure is part of the field of laser bio-printing (LAB), which aims to reconstruct human tissues in 3D. The LAB principle involves focusing a laser to create a plasma by absorption on a bio-ink film consisting of a solution of biomaterials, biochemical species and/or a cell suspension in a liquid medium. From this plasma, a cavitation bubble is generated in the bio-ink. This bubble, through its hydrodynamic movement, deforms the free surface of the bio-ink to the point of creating a stream of matter. The characteristics of this stream depend on a large number of physical parameters. It is through this stream (which contains a small number of cells, biomaterials or chemical species) that the transfer of material to the receiving substrate takes place in a controlled manner.

(5) In this context, it is necessary to print the constituents of biological tissues according to specific patterns/locations in order to obtain either 2D/3D objects that will have properties (shapes and functions) as close as possible to native living tissues, or chimeras allowing to render more complex, test, and/or simplify biological contexts to improve the understanding of tissue morphogenesis or biological response mechanisms to external agents (active agents).

(6) Thus, the printing of biomaterials carried out in conjunction with that of cells must also follow very specific printing schemes in order to provide a viable environment to the printed cells in order to achieve the requested item. In summary, controlling the quantity and position of cells, biomaterials, and/or biochemical species printed on the receiving substrate is essential to achieve the necessary and expected quality of bio-printed items.

(7) The purpose of the present disclosure is to set up a measuring device allowing identification and/or mapping of areas of specific composition in an inhomogeneous bio-ink before printing and thus trigger the laser to aim specifically at a desired composition area according to the area to be printed (on the “receiver”) and the pattern designed during the CAD of the target item.

(8) To this end, the invention comprises: i) carrying out a 2D characterization of the donor, ii) detecting inhomogeneities by ad-hoc digital processing, iii) automatically mapping the positions of specific composition areas, and iv) matching the laser shooting pattern (printing trajectory) with the mapping of areas of specific donor composition.

(9) In the context of bio-printing, particularly when developing new models, markers can be used temporarily to tag cells by external agents (immuno-marking, fluorescence, etc.) to assist in the characterization of printed items. On the other hand, when producing models for in vitro or in vivo applications, the cells must remain fully preserved from any exogenous disturbances because they constitute the building blocks of the target tissue. Therefore, they cannot be marked by external agents in these cases. Thus, the detection of cells will be relatively complicated within culture media because their refractive index is quite close to that of water, the main component of these media. Cellular imaging therefore already presents an important problem to be addressed.

(10) In addition, the filling dynamics of the printing head or the natural movement of the suspended cells have a direct impact on the position of the cells over time. This implies detecting their position repeatedly and at high frequency if the laser printing path is to be always in line with the mapping of their location. This has a direct consequence on the detection and mapping means which must operate at high speed (both for the hardware part allowing data acquisition and for the software part allowing data processing).

(11) The problem posed, which the present disclosure aims to solve, is therefore manifold and includes: to detect local variations in the composition of bio-inks (inhomogeneities) that are difficult to visualize (imaging means+image processing), to map inhomogeneities (image processing allowing both their number and position in the donor to be given), and to perform laser shots in correspondence with the mapping (adaptation of the printing trajectory in real time).

(12) According to a variant, the present disclosure also aims to cover very interesting cases of cell sorting: being able to distinguish between living and dead cells in order to print only living cells; being able to dissociate two cell types that would have been mixed (voluntarily by co-culture or not) in the same ink; being able to distinguish between cells of the same type but with a high degree of disparity (stem cells, different levels of differentiation/maturity); being able to distinguish between areas of concentrations of the same biomaterial that would be present in the observation area and produced voluntarily or not; being able to distinguish between different biomaterials that would have been mixed in the same bio-ink.

(13) At the performance level, the “Target-Shoot” function, object of this invention, may allow: the streamlining of the printing process (ratio of the number of printed cells to the number of cells present in the bio-ink), otherwise known as “Printing Yield.” This minimizes the number of cells not used during the process; the optimizing of the process by choosing laser shooting zones based on the number of cells spatially present (without the need to use multiple laser shots to print the same area). This means optimizing the printing path to minimize the number of laser shots and thus minimize cellular stress; speed up the process (if necessary) thanks to the efficiency of laser firing, otherwise known as “Printing Rate”; make the process compatible with a continuous recharging cartridge since the “Target-Shot” method must allow at any time to measure the cell displacements in the bio-ink where the flow will be relatively high.

(14) The stakes are also: to allow printing cells in small or rare quantities thanks to the mastery of the “Printing Yield.” This is an extremely important point in relation to certain pharmaceutical, cosmetic or clinical applications where the number of available cells is very low. to allow for a advanced customization of tissue models by the controlled choice of laser firing areas according to the number of cells spatially present. Thus, the response to requests for the development of custom-made tissues from laboratories or manufacturers will be possible with a very wide range of markets and applications, made possible by the high resolution and high precision of the laser process assisted by the Target-Shot method. to allow industrialization of the process and the increase in its productivity thanks to the acceleration of the process (“Printing Rate”) by the systematic efficiency of laser shots ensured by the Target-Shoot function. to make the use of the technology much easier because the handling of bio-inks will be optimized and automated with the continuous recharging cartridge. to control the number of particles transferred at any time during the bio-printing process (laser pre-print/laser post-print comparison) to ensure a good match between the printed pattern and the CAD-designed pattern. reduce the cost of bio-printing devices by integrating a single cartridge (or a reduced number of cartridges) into which the different tissue components that would be sorted at the time of transfer would be placed. Potentially, the cost reduction could even pave the way for the definition of a single-use cartridge that would be of great benefit for the use of bio-printing in the clinical field.

(15) In accordance with the present disclosure, an apparatus includes a camera (1) comprising a high-definition sensor, for example, of 18 megapixels. For example, the camera (1) is a sensor marketed under the trade name “USB 3 uEye CP” by the IDS company of Obersulm, Germany.

(16) This camera (1) is associated with a second image-combining optical unit (2) acting as a field lens and thus ensuring the combining of the image between the focal plane of the film (3) and the plane of the camera (1).

(17) The film (3) is placed in front of a target (11) to which the cells or particles are transferred when a laser pulse is triggered.

(18) For example, the second image-combining optical unit (2) consists of a lens, preferably telecentric, comprising at least two lenses optimized in the visible range.

(19) The optical path is reflected by a high-pass dichroic mirror (4), transmitting infra-red (corresponding to the emission wavelength of the laser (5)) and reflecting wavelengths in the visible range.

(20) The equipment also includes a light source (6) emitting in the visible range associated with a shaping optics (7) whose function is to collimate the light source (6) if necessary, for example, when the source is divergent from the emitted beam. This light source can be a single LED source, a component consisting of an array of LEDs, or a white light source such as incandescent lamps, halogen lamps, supercontinuum lasers, etc. The light source can also consist of a narrow spectrum source (either by the very nature of the technology used or by the use of optical filters) that emits in the wavelengths that allow the fluorescence excitation of markers or particles.

(21) A separator slide (8) is used to superimpose the lighting optical path and the observation optical path.

(22) At the exit of the dichroic mirror (4), the two beams heading toward the scanner (lighting and laser) are co-linear with each other and are in fact also co-linear with the imaging beam returning from the film. Thus, the three beams are co-linear between the dichroic mirror (4) and the ink film (3).

(23) They are deflected by a scanner (9) ensuring an orientation that is controlled by an external computer.

(24) The scanner (9) provides an angular orientation of the three co-linear beams mentioned above, along two perpendicular axes, two of which beams are scanned on the donor containing the bio-ink and the last being “deflected” to a collimated imaging beam toward the imaging system. The scanner (9) includes, for example, two mirrors driven by an electromagnetic actuator, for example, one marketed by SCANLAB Company of Puchheim, Germany, under the trade name “SCANcube 14.”.

(25) The three observation, lighting and laser beams are thus co-linear and oriented in the same direction as the aperture of the scanner (9). Thus, the observation direction and the lighting direction follow the orientation of the laser beam.

(26) The function of the optical unit (10) is to: a) transform the angular orientation into a lateral positioning/displacement along two axes X,Y in the plane of the film (3); b) focus the laser beam and the lighting beam in the same plane of the film (3); and c) collect the light in the visible domain reflected by the film (3), to construct the observation image of the camera (1).

(27) The optical unit (10) comprises of a set of lenses forming a telecentric lens with the following characteristics:

(28) In the infra-red spectrum, the lens surfaces are treated with anti-reflective coatings to support high laser energies. This prevents the deterioration over time of the first optical unit (10), the design of which is calculated to prevent the creation of laser “hot spots” within the first optical unit (10).

(29) The dichroic mirror (4) prevents the return of laser infra-red radiation to the camera (1) when a pulse is triggered. Optionally, an infra-red rejection filter can also be placed in the optical path between the dichroic mirror (4) and the camera (13).

(30) The ratio of the focal length of the second image-combining optical unit (2) and the focal length of the optical unit (10) is determined to provide in the plane of the camera (1) an image whose smallest observed objects have a size of more than one pixel.

(31) The equipment also includes a computer (12) receiving data from the camera (1) and a second camera (13) observing the target (11). This computer (12) also controls the scanner (9) and the laser (5).

(32) The computer (12) runs computer programs to perform different processing tasks: the pre-processing of the raw images from the camera (1); a localization and characterization processing of the particles identified during the aforementioned pre-processing; a control processing of the scanner (9) and the laser (5) to match the laser's shots with the position of the particles; and optionally a processing of the raw images from the camera (13) observing the target (11).

(33) The raw images from the camera are pre-processed. This pre-processing can be carried out periodically, to record a mapping of the film (3) before a laser triggering sequence is engaged, or between two consecutive triggering sequences.

(34) The first solution is particularly suitable for situations where the film carries stable particles, both in terms of their location in the film plane and in terms of their development. This concerns, for example, mineral or organic particles that are not very reactive with respect to the substrate.

(35) The second solution is better suited to situations where the particles are mobile and scalable. This is the case, for example, of living particles such as cells, which can move in the film with a strong tendency to aggregate that will strongly depend on the type of cell and medium used.

(36) In both cases, the processing consists in extracting information from the raw images corresponding to the detection of graphic items corresponding to particles of interest, for example, by segmentation, shape recognition, or contour and center of gravity recognition techniques.

(37) Watershed, Meyer's flooding algorithm or Optimal spanning forest algorithms thresholding techniques may be used to perform this mapping.

(38) This processing can be divided into several phases: a phase of restoration or conditioning of the raw images (noise suppression, de-blurring, correction of non-uniform lighting); a segmentation phase; a characteristic extraction phase; and a data analysis phase.

(39) This processing assigns to each identified graphic item an ID± identifier and a position in the form of Cartesian coordinates in the film plane.

(40) FIG. 2 shows the sequence of images, based on the raw image (20) captured by the camera (1), and an enlarged area (21) showing the presence of a plurality of particles in the image field.

(41) A thresholding processing is used in a known manner to calculate a contrast variation in the form of a histogram, as shown in the image (22). This histogram allows calculation of a contrasted image (23) by using thresholding algorithms.

(42) This contrasted image (23) is used to calculate the centroids (image (24)) and contours (image (25)).

(43) This information makes it possible to build the table (26) of the identifiers of each processed graphic item and the coordinates of the center of each of these items.

(44) The characterization processing consists in assigning attributes to each of the identified and localized graphic items according to their affiliation to a family predefined by these physical parameters such as: the size class of the item reaction to specific lighting, e.g., photoluminescence spectral characteristics the type of contour (shape, regularity, etc.) the affiliation to an aggregate of items the optical density of the item the density of the aggregate (estimated number of aggregated items) spatio-temporal dynamics

(45) These parameters are of course not limiting.

(46) The result of this processing completes the above table by adding to each identifier information about the affiliation to one or more series of classes.

(47) Processing is preferably made in parallel and run on parallel architecture processors, such as GPUs.

(48) The information is then used to match the mapping thus carried out with the previously recorded target map in order to calculate a sequence of laser shots resulting in the calculation of a scanner orientation and then the triggering of the laser pulse, under the control of the computer (12). The calculation of the sequence takes into account a calculation of the reduction of the global process time, by known algorithms such as “resolution of the commercial traveler's optimization problem or of the NP-complete problem.”.

(49) After each laser shot or after a series of laser shots, the computer checks the conformity of the transfers by comparing the image transmitted by the camera (13) observing the target (11) and the pre-registered target map. The calculator performs a maximum likelihood calculation, and in case of a discrepancy (lack of printed items), recalculates the following sequence of laser shots to correct the observed anomalies. It can also be used to correct areas where there have been too many printed items with respect to the target map. This correction is generally done by conventional suction means that allow very precise removal of small quantities of material. This suction is controlled by the computer (12). In any case, control here means the installation of a control loop between the pre-recorded target map and the target (11) actually printed, which must make it possible to obtain a maximum likelihood as close as possible to 100% between both maps.

(50) Moreover, the present disclosure is not limited to a single laser printing process. It covers multi-head printing (several films (3) used at the same time with one or more lasers), for which it is imperative that the imaging means and their associated processing be capable of processing several areas simultaneously in order to guarantee the possibility of simultaneous “multicolor” printing (several different cellular types printed in a single process).